**3. NP entrapping various Ags for delivering cancer vaccines**

cytosolic pathways have been identified to utilize endosomes and endoplasmic reticulum, respectively, to generate the MHC-I/Ag peptide complexes [20–22]. Notably, the endosome pathway in normal conditions is devoted to process the exogenous Ags to form the MHC-II/ Ag peptide complexes for activation of CD4+ T cells, although the abnormal increase in endosomal pH or occurrence of endosome breakup made purposely by artificial strategies is thought to prevent the protease-mediated degradation of Ags in endosomes, thus promoting cross-presentation [12, 23], and additionally, certain DC subsets such as tissue-resident CD8+ and migratory CD103+ DCs in mice and CD141+/BDCA-3+ DCs in humans were reported to

After activation and differentiation from CD8+ T cells in lymphoid tissues, the matured Ag-specific cytotoxic CD8+ T lymphocytes (CTLs) enter the systemic circulation and patrol peripheral tissues in search of target cells, which display a specific Ag epitope in the context of MHC-I matching the Ag-specific T cell receptors (TCRs) on CTLs, which once identification of the target cells will secrete perforin and granzymes to lyse them and within minutes move on to kill the next target [26]. By contrast, CD4+ T cells mainly play a helper role of regulation of immune responses as manifested by the observations that after activation by MHC-II/Ag peptide complex presented by DCs, naïve CD4+ T cells differentiate into four distinctive subtypes depending on the polarizing cytokines [27]. Type 1 helper T cells (Th1) induced by IL-12 secrete IL-2 and IFN-γ to promote CD8+ T cell responses; Th2 cells induced by IL-4 secrete IL-4 and IL-5 and are involved in humoral immune responses; regulatory T cells (Tregs) induced by IL-2 and TGF-β (transforming growth factor beta) secrete TGF-β and IL-10 to suppress immune responses; and Th17 cells induced by TGF-β, IL-6, and IL-21 secrete IL-17 and IL-22 to break immune tolerance and possibly leading to autoimmunity [27, 28]. In addition, it is reported that CD4+ helper T cells are utilizing the expressed CD40L for feeding back to DCs to further amplify immune activation and aid in establishment of memory CD8+ T cell responses [29, 30]. To prevent cancerous occurrence, the immune system constantly implements a process referred to as immunosurveillance whereby to inhibit oncogenesis by actively identifying and eliminating tumor cells, which however, have also devised mechanisms to evade immune responses, including downregulation of tumor Ags and promotion of immunosuppression [31, 32]. In established tumor microenvironment, it is generally immunosuppressive due to upregulation or production of inhibitory molecules, such as TGF-β1, CXCL12, VEGF, ARG1 (Arginase1), CCL18, iNOS (nitric oxide synthase), IL-10, IL-35, and galectin-1 by many types of cells, including cancer-associated fibroblasts, myeloid-derived suppressor cells, Tregs, and tumor-associated macrophages (TAMs), against T cells [33]. Also, activated T cells upregulate CTLA-4 (CTL-associated protein 4) which binds to co-stimulatory molecules on DCs with higher affinity than CD28, serves as a peripheral inhibitory signal to prevent over-reactivity of T cells, and dampens antitumor immune responses. Besides, tumor cells can also secrete cytokines such as IL-10 and TGF-β, which both directly inhibit the proliferation of CTLs and drive the differentiation of Tregs to provide an additional source of immunosuppressive cytokines, while subsets of tumor cells highly express programmed death-ligand 1 (PD-L1) for binding to programmed death-1 (PD-1) on T cells and inhibiting their effector functions [34]. Thus, tumor cells can promote immunosuppressive tumor microenvironment and shield themselves from CTLs by hijacking normal negative feedback loops designed to guard against

be more efficient at Ag cross-presentation than other DC subtypes [24, 25].

50 Immunization - Vaccine Adjuvant Delivery System and Strategies

Tumor Ags include mutated cell surface components, such as polysaccharides, peptides, oncoproteins, and DNA and mRNA that encode those proteins, which as referred to subunit Ags, meanwhile tumor cell lysate and immunogenically dying tumor cells can also serve as the source of whole-cell Ags [6]. As key components utilized for formulating anticancer vaccines, subunit Ags have major advantages including defined chemical synthesis; ease of production; and for vaccine formulations, requiring, possibly, no Ag-processing by APCs and challenges including elicitation of humoral rather than cellular immune responses, poor delivery efficiency, and in vivo stability. Whole-cell Ags have major advantages including broad-epitope immune responses, potential for "personalized" therapy, full preservation of tumor Ags and challenges including production requiring tissue biopsy, difficulty in manufacturing, loss of antigenicity during production, presence of self-Ags, and immunosuppressive molecules such as PD-L1. Notably some viruses, such as Epstein–Barr virus (EBV), human papilloma virus (HPV), and hepatitis B and C viruses, have proven to contribute to certain cancer-related development, and therefore, their virally gene encoded surface proteins may also serve as the potential target Ags to constitute the vaccines for cancer immunotherapy [35, 36]. Among different types of tumor Ags, oncoproteins, which are encoded by oncogenes involved in the regulation or synthesis of proteins linked to tumor cell growth and may also be either mutated or overexpressed normal or embryonic proteins from fetal development, are intensively investigated for cancer vaccines since they have a big potential in induction of broad-epitope CD8+ and CD4+ T cell responses. Notably, compared to full-length protein-based Ags that require cellular uptake and processing for presentation to T cells, peptide epitopes can directly bind to MHC molecules and thus directly activate T cells and, moreover, are more endurable to damages during the preparation and storage of vaccine products, thus, in line with these advantages, leading to many ongoing clinical trials on peptide-based cancer vaccines [37, 38].

However, poor immunogenicity and limited therapeutic efficacy are still big challenges in developing protein, especially, peptide Ag-based subunit vaccines that are designed for cancer immunotherapy; for example, in the case of melanoma, the identified Ags include β-catenin, survivin, tyrosinase, gp100, MAGE, melan-A (MART1), and NY-ESO-1, some of which, such as gp100 and MAGE-A3 peptides, when tested in clinical trials just showed only moderate or null therapeutic efficacy [39]. Grooming through clinical trials on peptide-based cancer vaccines, it may be safely concluded that therapeutic efficacy of subunit vaccines against cancer remains suboptimal [2], due to at least partially the fact that many tumor Ags evaluated in clinical trials are self-Ags which can hardly trigger the autoreactive T cells leading to immunotolerance [6]. These disappointed outcomes highlight that the conventional subunit vaccines should actually be formulated with innovative modalities, which may be an alternative promising strategy to further improve cancer immunotherapy, as evidenced by positive results obtained from pre- and clinical investigations carried out more recently on cancer vaccines that were combined with other elements, such as potent adjuvants and NP-based VADSs. For example, in a preclinical study, researchers observed that, in a syngeneic mouse model of oral cancer comprised of mouse tonsil-derived epithelial cells stably expressing HPV-16 E6 and E7 genes along with H-ras oncogene (mEER), intranasal HPV E6/E7 peptide vaccination or single checkpoint antibodies failed to elicit responses in most mice; however, 4-1BB agonist antibody along with either CD40 agonist antibody or CTLA-4 blockade eliminated the majority of established mEER tumors, and even produced a curative efficacy and a high safety profile against orally implanted mEER tumors [40]. For another example, in a phase II clinical trial, researchers performed immunotherapy with two peptide cancer vaccines in combination with intravesical bacillus Calmette-Guerin (BCG) for patients with non-muscle invasive bladder cancer (NMIBC) and demonstrated that this combinatory immunotherapy had good immunogenicity and safety and resulted in a 2 year RFS rate 74.0% in all patients, suggesting the cancer vaccines with a combinatory mode may provide benefit to patients for preventing recurrence of NMIBC [41].

for cross-presentation and favorably elicit CD8+ T cell responses [24]. As such, to engender Ag lysosome escape, great efforts have been focused on pH-sensitive delivery systems that can retain the loaded cargo under the physiological pH condition while triggering release of Ags and disruption of endocytic vacuoles at the acidic (below pH 6) endosomal microenvironment [42], as exemplified by a pH-sensitive liposomal VADS which is formulated with a

More recently, Wang and colleagues through fabricating two types of pH-sensitive multifunctional liposomes, the mannosylated lipid A-liposomes (MLLs) and the stealth lipid

proSLL/MLL-constituted microneedle array (proSMMA), which dissolved rapidly recovering the initial MLLs and SLLs upon rehydration [12]. Mice vaccinated with proSMMAs by vaginal mucosa patching elicited robust Ag-specific humoral as well as cellular immunity at both systemic and mucosal levels, especially, in the reproductive and intestinal ducts. Further exploration revealed that the Ags delivered by either liposomes were cross-presented for MHC-I displaying by APCs thanks to lysosome escape and reactive oxygen species stimulation, both

ammonia induction, resulting in a mixed Th1/Th2 type response which was also promoted by liposomal lipid A via activation of TLR4, indicating the proSMMAs a multifunctional VADS capable of engendering Ag lysosome escape to elicit robust humoral and cellular immunity

In addition, an alternative approach for evading lysosome degradation of Ag includes multifunctional VADS constituting of the oxidation-sensitive polymersomes that can respond to the oxidative environment of endosomes and deliver Ags and adjuvants to cellular cytosol for induction of cellular immune responses [44]. Notably, liposomes modified with a cellpenetrating peptide octaarginine were also reported to be able to promote cross-presentation Ags and elicit production of anticancer CTLs, because the membrane-penetrating liposome enhanced proteolysis of the exogenous Ags by proteasomes and amino peptidases facilitating promoting the C-terminal trimming of antigen peptide and the production of mature MHC-I peptides [45]. Also, gold nanoparticles displaying tumor Ags were reported to enable efficient antigen delivery to dendritic cells and then activate the cells to facilitate cross-presentation,

Recently, the approach based on amphiphilic polymer-Ag peptide conjugates through the conjugation of azide-functionalized Ag peptides to an alkyne-functionalized core via azidealkyne click chemistry has been employed for making nanovaccines against cancer. For example, by conjugation of the melanoma Ag peptide TRP2 and azido PEG mannose to the alkyne polymer, an anti-melanoma nanovaccine with the size of 10–30 nm was formed via self-assembly and was efficiently taken up by DCs [47]. In spite of poor immunogenicity, when given to model mice with B16-F10 melanoma tumors together with the adjuvant CpG, the adjuvanted TRP2-nanovaccines effectively suppressed the tumor growth and significantly

against Ags and a promising platform for making both cancer and infection vaccines.

inducing Ag-specific CTL responses for effective cancer immunotherapy [46].

**4.2. NPs targeting DC for delivering cancer vaccines**

HCO<sup>3</sup>

to rupture lysosomes by gas expansion and to cause ROS production by excessive

, into microneedles prepared the

Vaccines Developed for Cancer Immunotherapy http://dx.doi.org/10.5772/intechopen.80889

HCO<sup>3</sup>

into CO<sup>2</sup>

and

53

dextran derivative and was shown to promote cytosolic delivery of Ags [43].

of which occurred when lysosomal acidifying the liposome-released NH4

A-liposomes (SLLs) both loaded with Ags and NH<sup>4</sup>

NH4 + /NH<sup>3</sup>

These investigations showed that the conventional vaccines have limited capability to target delivery of tumor Ags and adjuvants to proper APC and intracellular compartments and may be renovated by the NP-based vaccine adjuvant-delivery systems (VADSs) which have already poised to address these challenges as described below.
